The invention relates to peptide inhibitors of glycosaminoglycans. This invention also relates to formulations, uses and methods of identifying such inhibitors.
The extracellular matrix (ECM) is a dynamic assemblage of interacting molecules that regulate cell functions and interactions in response to stimulation. One class of extracellular matrix macromolecules, the glycosaminoglycans, are molecules known to be involved in a wide array of both normal and abnormal biological processes, including cell migration, differentiation, proliferation, immune response and cytoskeletal organization.
The glycosaminoglycan hyaluronan (HA) is a repeating disaccharide of [GlcNAcxcex21-4GlcUAxcex21-3]n that exists in vivo as a high molecular weight linear polysaccharide. HA is found in mammals predominantly in connective tissues, skin, cartilage, and in synovial fluid, and is also the main constituent of the vitreous of the eye. In connective tissue, the water of hydration associated with HA creates spaces between tissues, thus creating an environment conducive to cell movement and proliferation. HA plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, embryological development, wound healing, angiogenesis, and tumorigenesis (Toole, Cell Biol. Extracell. Matrix, Hay (ed), Plenum Press, New York, 1384-1386 (1991); Bertrand et al. Int. J. Cancer 52:1-6 (1992); Knudson et al, FASEB J. 7:1233-1241 (1993)). HA levels have been shown to correlate with tumor aggressiveness (Ozello et al., Cancer Res. 20:600-604 (1960); Takeuchi et al., Cancer Res. 36:2133-2139 (1976); Kimata et al., Cancer Res. 43:1347-1354 (1983)), and can be indicative of the invasive properties of tumor cells. M. M. Knupfer et al., Anticancer Res 18:353-6 (1998).
HA is also involved in immune response, and thus may mediate this response in both normal and abnormal biological reactions. Increased binding of HA to one of its receptors, CD44, has been shown to mediate the primary adhesion (xe2x80x9crollingxe2x80x9d) of lymphocytes to vascular endothelial cells under conditions of physiologic shear stress, and this interaction mediates activated T cell extravasation into an inflamed site in vivo in mice. H. C. DeGrendele, et al., J. Exp. Med. 183:1119-1130 (1996); H. D. DeGrendele,et al., Science 278:672-675 (1997). H. C. DeGrendele et al., J. Immunol. 159:2549-2553 (1997). Alterations in levels of HA and other glycosaminoglycans have also been associated with unwanted immune responses, and may be involved in diseases and disorders such as rheumatoid arthritis, atopic dermatitis, psoriasis, multiple sclerosis, transplantation rejection. For example, HA and other glycosaminoglycans display are altered in autoimiune disorders such as arthritis, and decreased levels of both hyaluronic acid and chondroitin 6-sulfate have been found in the diseased synovial fluid of both adults with rheumatoid arthritis (A. Bensouyad et al., Ann Rheum Dis 49:301-7 (1990)) and children with juvenile rheumatoid arthritis (P. F. Spelling et al. Clin Exp Rheumatol 9:195-9 (1991)).
Dendritic cells (DC) play essential roles in the induction of cellular immune responses to a variety of relevant antigens. DC are known to play critical roles in the induction of cellular immune responses against a wide variety of antigens of relevance, including chemical haptens, foreign proteins, infectious microbes, and tumor-associated antigens (Steinman, xe2x80x9cThe dendritic cell system and its role in immunogenicity.xe2x80x9d Ann. Rev. Immunol. 9:271 (1991); Stingl et al., xe2x80x9cThe Epidermis: An Immunologic Microenvironment In Dermatology in General Medicine.xe2x80x9d T. B. Fitzpatrick, ed. McGraw Hill and Co., New York, p. 172 (1993)). Interaction between HA, expressed on endothelial cells, and CD44, expressed on activated dendritic cells as well as T cells, and granulocytes, is believed to mediate homing of such leukocytes to their target sites.
Glycosaminoglycans, and particularly HA, are also known to mediate other cellular interactions that involve binding and entry into a cell. For example, HA is involved in infection of mammalian cells by the Human Immunodeficiency Virus (HIV), since HIV is known to bind to HA upon infection. Both HA and monoclonal antibodies to its receptor CD44 were found to inhibit HIV infection of monocytes by monocytotropic HIV. M. C. Levesque and B. F. Haynes, J. Immunol 156:1557-65 (1996). HA is also involved in mammalian zygote formation by mediating binding of the oocyte and the sperm. Data suggests that HA in the cumulus matrix may act to prime the fertilizing sperm for induction of the acrosome reaction by constituents of the cumulus and/or zona pellucida. HA is thought to mediate this interaction by binding to the PH-20 protein to increase basal levels of intracellular calcium and thereby potentiate the acrosome reaction. K. Sabeur et al., Zygote 6:103-11 (1998). HA mediates sperm motility by enhancing phosphorylation of proteins including HA binding protein. S. Ranganathan et al., Cell Mol Biol Res 41:467-76 (1995).
The role of glycosaminoglycans, and particularly hyaluronic acid, in such varying physiological processes make them attractive targets for therapeutic agents. Unfortunately, glycosaminoglycans have been found to be nearly non-antigenic, and very few antibodies that recognize glycosaminoglycans have been isolated. Due to this lack of antigenicity, it has been technically difficult to develop inhibitors or probes of glycosaminoglycans. Thus, there is a need in the art for inhibitors of glycosaminoglycan-mediated processes, and in particular for inhibitors of hyaluronic acid-mediated processes. There is also a need for a method of identifying effective glycosaminoglycan inhibitors in a systematic, reproducible manner.
The present invention provides peptides with a specific affinity for glycosaminoglycan molecules. These peptides exhibit any number of functions, including but not limited to inhibitors of glycosaminoglycan-mediated processes, enhancers of glycosaminoglycan-mediated processes, and as molecular probes to identify the presence of a specific glycosaminoglycan. Peptides of the invention are administered in order to inhibit, alter the interaction of, or otherwise affect the activity or function of any glycosaminoglycan, including hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, heparin, keratan sulfate, keratosulfate, chitin, chitosan 1, and chitosan 2. These isolated peptides are formulated and administered by injection for the treatment or prevention of diseases involving viral infection, inflammatory diseases, cancer, infections, etc. Moreover, these peptides are formulated and administered for the stimulation of normal biological responses, such as wound healing, angiogenesis, etc. The peptides of the invention cab be labeled directly or indirectly and as such are useful in in vitro, ex vivo or in vivo probes to determine the presence of a particular glycosaminoglycan in a biological sample and/or patient.
In a preferred embodiment, the invention provides isolated and substantially purified peptides which specifically bind to and as such inhibit or otherwise affect the activity of HA. The isolated peptide inhibitors are characterized by aliphatic or polar aliphatic residues at positions 4, 5 and 6 and/or 9, 10 and 11.
An aspect of the invention is a composition comprising a carrier material and an active ingredient. The active ingredient is characterized by all or any of (1) specifically and selectively binding to a glycosaminoglycan which is preferably hyaluronic acid; (2) inhibiting the normal function of or altering the normal interactions of or otherwise affecting the normal activity of a glycosaminoglycan; (3) having an amino acid sequence corresponding to any of SEQ ID NOS:1, 2, 3, 4 or 5; or (4) having sufficient homology with any of SEQ ID NOS:1, 2, 3, 4 or 5 so as to present a structure characterized by (1) or (2) above.
The invention further provides pharmaceutical and cosmetic compositions containing a peptide that specifically modulates a glycosaminoglycan-mediated activity. The peptides in the pharmaceutical composition are used in conjunction with an acceptable pharmaceutical carrier, to prepare medicinal compositions for the treatment of glycosaminoglycan-mediated disorders in animals, and more preferably mammals, including humans. In a preferred embodiment, the glycosaminoglycan modulated is HA. In a more preferred embodiment, the peptide is an HA inhibitor comprising the sequence of SEQ ID NO:1.
The invention further provides the use of the described isolated peptides in cosmetic compositions, e.g. a topical skin cream, with an acceptable cosmetic carrier. Such topical skin creams may contain additives such as emollients, moisturizers, fragrance, and the like. In a preferred embodiment, the glycosaminoglycan modulated is HA, and the peptide used to modulate the activity is characterized by aliphatic or polar aliphatic residues at positions 4, 5 and 6 and/or 9, 10 and 11. In a more preferred embodiment, the peptide is an HA inhibitor comprising the sequence of SEQ ID NO:1.
The present invention also provides a method of blocking cell migration using peptides with specific affinity for glycosaminoglycan molecules. In one embodiment, the invention provides a method for preventing DC migration from the epidermis by blocking HA-CD44 interaction with a peptide inhibitor of HA. In a preferred embodiment, the peptide used to block the HA-CD44 interaction is comprised of SEQ ID NO:1.
The present invention also provides a method of preventing the induction of immune responses by altering the interaction of glycosaminoglycans and cell receptor molecules. Peptides specific for a glycosaminoglycan can inhibit interaction with cell surface molecules of migratory cells, thus inhibiting the migration process. In one embodiment, peptides specific for HA can inhibit the migration of immune cells by inhibiting interaction with CD44. In another embodiment, peptides that are specific for heparin can inhibit the migration of cells where the migration is dependent on the fibroblast growth factor receptor.
The present invention also provides a method of identifying peptides that specifically bind to a carbohydrate, such as a glycosaminoglycan, using a phage display technology. The method of the invention includes a step of selecting peptides that specifically bind to the carbohydrate of interest by collecting only the clones released from the carbohydrate-coated plates after treatment to neutralize the function of the carbohydrate, e.g. by addition of the same or similar carbohydrate in excess, by enzymatic or non-enzymatic digestion of the carbohydrate, by use of a chelator, and the like.
One aspect of the invention is a composition comprising a pharmaceutically acceptable carrier and an active ingredient have an amino acid sequence defined by a motif ZZZXZZZ where Z is either an aliphatic or polar aliphatic amino acid and X is any amino acid.
One feature of the present invention is that the peptides identified using the method of the invention inhibit glycosaminoglycan-protein interactions by binding to the glycosaminoglycan rather than to the protein with which it interacts.
Another feature of the peptides of the present invention is that they afford better inhibition of glycosaminoglycan-mediated activity than larger, less specific glycosaminoglycan inhibitors such as receptor antibodies, e.g. anti-CD44 antibodies.
An advantage of the peptide modulators of the present invention is that they are more specific than chemical inhibitors of glycosaminoglycans, e.g. tunimycin and H7.
Another advantage of the peptide modulators of the invention is that they are significantly smaller than other inhibitors of glycosaminoglycan-mediated activity, e.g. antibodies or soluble glycosaminoglycan. The smaller size of the peptide allows for better oral and topical formulations.
Another advantage of the present invention is that the peptide inhibitors of the invention is that they are more cost effective to produce than presently available modulators of glycosaminoglycan-protein interactions, e.g. CD44 inhibitors such as anti-CD44 antibodies.
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the presently described invention.